| Proteins play a key role in countless physiological processes in the human body. Many of these physiological processes are regulated on the cellular level by proteins such as Calmodulin (CaM). CaM is a calcium-binding protein that is found in the cytoplasm of eukaryotic cells. It acts as the primary mediator of Ca2+ and as a secondary messenger in calcium signal transduction pathways [Lu, 1993 #365]. CaM is a small, acidic protein composed of 148 residues and contains two globular domains (an N- and C-terminal domain) that are connected by a flexible tether [Babu, 1988 #473].; The goal of the research was to monitor the effects of calcium binding on the stability of the C-domain of Calmodulin (CCaM) representing this through a simple thermodynamic cycle (Figure below). In order to assess this linkage, we simplified our model from a 4-site system (CaM; whole Calmodulin) to a 2-site system (CCaM; isolated C-domain of CaM), and then a 1-site system (mutants of the C-domain). Simplifying our system excluded interdomain interactions between the N- and C-domains as well as cooperative calcium binding within a domain. This cycle illustrates the linkage between unfolding of the apo- and saturated-native states and the binding of calcium to the native and unfolded states.*; This thesis describes the linkage of Ca2+ binding to the stability of the protein, the assessment of the intrinsic Ca2+ binding constants and the intradomain cooperativity for multi-site proteins. These properties are determined experimentally for the wild-type CCaM and the mutants of CCaM in order to understand the factors that influence the relationships within this linkage. We found that many factors such as ionic strength or salt effects, temperature and types of denaturants, etc. play a significant role in perturbing the system of the C-domain mainly because of the acidic nature of the protein. Attempts to account for all factors, which influenced our results and the interpretation of those results, were addressed through models accounting for the equilibria of each leg of the cycle.{09}Through data analysis of our Ca2+ binding and stability studies of CCaM and its mutants, we observed a linkage, but one that is not well represented by a thermodynamic box but a triangle. Thus, Ca2+ binding to the unfolded state does not contribute significantly to the energetics of Ca2+-induced stability of CaM.; *Please refer to dissertation for diagrams. |
